基于角膜电氛的氨基葡聚糖类双重效应囊泡衣膜的构建及其促透作用研究

Corneal epithelia alongwith the tear turnover, serve as biological barriers for the entrance of extraneous substance for topical ocular administration. The use of surface modified colloidal drug delivery system is a suitable strategy to obtain enhanced bioavailability. However, the exhibition of reduced precorneal drug loss and improved corneal permeability for modified nanocarriers and the related influence of corneal charges, and the prediction of behavior profile for nanocarriers in targeted tissue are still undetermined. Our research group found that some glycosaminoglycans exhibited a higher permeation into deeper corneal layers, especially with longer contacting time because of corneal charges. Therefore, taking advantage of the high durg encapsulation efficiency and sustained release characteristics of liposomes, we chose chitosan, chitosan oligosaccharide, N-trimethyl chitosan chloride and N-carboxymethyl chitosan as vesicle coatings according to charges, and applied curcumin and cyanidin capturing free radicals as model drugs with low permeability according to water-solubility. With the construction of novel glycosaminoglycan-coated liposomal drug delivery system, after the evaluation of dual function and the mechanism, we are assumed to guide the design of surface modified nanocarriers with the help of transmission rule interpreting the interaction of coating, lipid, drug, drug delivery system, and biological effect. Furthermore, the versatile and systematic research pattern is supposed to be established for the construction and application of macromolecule coatings, improving the theory of pharmaceutics in ophthalmic drug delivery system.

角膜前难滞留及角膜脂水层交替结构是药物眼前段吸收的主要屏障，研究膜修饰纳米载体是促进药物眼部吸收的热点。然而修饰后载体是否降低药物眼前清除率或/并促进其跨膜吸收、角膜电氛在诱发该双重效应中的角色及机理、制剂在靶组织的行为预测是亟待解决的关键科学问题。本课题组研究发现某些氨基葡聚糖类在角膜电氛环境下发生生物黏附，并显著促进微粒以细胞旁路途径渗透至角膜上皮细胞深层。结合脂质体对药物的高效包封及缓慢释放，本课题以电荷为标准，选择壳聚糖、壳寡糖、三甲基壳聚糖、羧甲基壳聚糖为衣膜材料；以水溶性为标准，选择具有捕获自由基且跨膜吸收障碍特征的姜黄素、矢车菊素为模型药物，通过构建新型衣膜修饰脂质体给药系统、总结衣膜-脂质-药物-给药系统-生物学效应传变规律，对衣膜双重促透作用及机制进行研究，以期指导设计膜修饰纳米载体，为高分子衣膜的构建及应用提供通用性系统性的研究模式，进一步完善眼部给药系统的制剂学理论。

角膜前难滞留及角膜脂水层交替结构是药物眼前段吸收的主要屏障，研究膜修饰纳米载体是促进药物眼部吸收的热点。然而修饰后载体是否降低药物眼前清除率或/并促进其跨膜吸收、角膜电氛在其中的角色及机理、制剂在体行为预测是亟待解决的关键科学问题。为解决以上问题本课题组以电荷为标准，选择壳聚糖、壳寡糖、三甲基壳聚糖、羧甲基壳聚糖为衣膜材料；以水溶性为标准，选择具有捕获自由基且跨膜吸收障碍特征的姜黄素，矢车菊素为模型药物，构建新型衣膜修饰脂质体给药系统。分别以乙醇注入法、逆向蒸发法制备GLDDS脂质体，并完成衣膜材料的合成和修饰，测定修饰脂质体理化性质、膜流动性、FALT最终构建制剂稳定性良好、符合眼用制剂学标准的GLDDS系统。采用微渗析结合HPLC/MS手段，考察GLDDS在家兔眼部房水中的药动学行为发现，两种药物的AUC0-t和MRT均显著提高，其中以荷正电的TMC包覆纳米囊泡作用最强。各系统对不同水溶性药物的角膜促透作用及作用机理通过放射性标记、荧光染料标记，采用非损伤性γ-闪烁示踪显像技术、黏蛋白混合检测、离体兔角膜透过实验及共聚焦激光扫描显微镜成像技术进行研究。结果显示TMC对脂质体的在体黏膜黏附性作用最强，与黏蛋白Ⅱ类作用显示同样趋势。采用荧光示踪，发现氨基葡聚糖类可显著促进微粒以细胞旁路途径渗透至角膜上皮细胞深层45-60 μm。根据GLDDS对紧密连接膜蛋白Occludin蛋白表达结果显示，这可能是囊泡荷电表面与紧密连接处相互作用导致的渗透增加。当氧化损伤细胞经GLDDS制剂孵育后，测定细胞活力、形态、早期凋亡、胞内ATP含量及线粒体膜电位等结果显示，除Cur混悬液组外，与普通脂质体组比较，细胞抗氧化损伤能力均有所提高，其中以TMC包覆制剂效果最明显。建立硒性白内障大鼠模型，晶状体浑浊度及相关酶活力测定结果显示，与模型组相比，给药组白内障可得到明显延缓，生化指标改善，其中TCL效果明显，OCL及CL间无显著差别。通过数学模型相关分析方法，结果显示对于溶解性相似的药物来说，GLDDS可同化模型药物理化性质，使最终制剂更多地显示衣膜优势，不同衣膜理化性质中以黏附性、电位、FALT值与体内生物效应正相关性最显著。本结论可为高分子衣膜的构建及应用提供通用性系统性的研究模式，进一步完善眼部给药系统的制剂学理论。